Protein Protocols Protein Protocols

AUT Gel for Histones 119
13. Add 0.05 mL of sample buffer per sample tube with lyophilized protein to be analyzed
in one gel lane (see Note 9). Add 1 mL of sample buffer to sample for one
gradient gel. To assure full reduction of all proteins by DTT, the pH must be above
8.0. If the pink phenolphthalein color disappears because of residual acid in the
sample, a few microliters of concentrated ammonium hydroxide should be added to
reach an alkaline pH.
14. Limit the time for sample solubilization and reduction to 5 min at room temperature to
minimize the possibility of protein modification at alkaline pH by reactive urea side reactions,
for example, by modification of cysteine residues by cyanate.
15. Acidify the sample by addition of 1/20 volume of glacial acetic acid.
16. Add methylene blue running front dye: 2 µL per gel lane or 50 µL for a gradient gel.
17. For a regular AUT gel, prepare reference histone samples: To 2 and 6 µL reference histone
solution with 10 and 30 µg of total calf thymus histones, one adds 40 µL sample buffer
(step 12), 2.5 µL of glacial acetic acid, and 2 µL of methylene blue.
18. When stacking gel polymerization is complete, remove the comb. Drain the wells completely,
using a paper tissue as wick, to remove residual unpolymerized gel solution. At
comb and spacer surfaces, gel polymerization is typically incomplete. The high urea concentration
of unpolymerized gel solution interferes with the tight application of samples.
19. Remove the bottom spacer from the gel assembly gel assembly and use it to remove any
residual Vaseline from the lower surface of the gel.
20. Clamp the gel assembly into the electrophoresis apparatus and fill the lower buffer reservoir
with electrophoresis buffer.
21. Use a 5-mL syringe with a bent syringe needle to displace any air bubbles from the bottom
of the gel.
22. For regular gel, follow steps a–c and continue at step 24.
a. Samples are applied deep into individual sample wells by Hamilton microsyringe
(rinsed with water between samples) (see Note 10). For the combination of comb and
gel dimensions listed, a 50-µL sample will reach a height of 1 cm (see Note 7). Samples
can also be applied to sample wells by any micropipetter with plastic disposable tip.
Pipet each sample solution against the long glass plate and let it run to the bottom of
the well.
b. Apply reference samples in the outer lanes, which frequently show a slight loss of
resolution due to edge effects. The threefold difference in reference protein amounts
facilitates correct orientation of the gel following staining and destaining and obviates
the need for additional markings. Optionally, apply 50 µL of acidified sample buffer to
unused lanes.
c. Gently overlayer the samples with electrophoresis buffer, dispensed from a 5-mL
syringe fitted with a 21-gauge needle until all wells are full.
23. For gradient gel, follow steps a–c and continue at step 24.
a. Fill the block well with electrophoresis buffer.
b. Use a level to confirm that the bottom of the preparative well is exactly horizontal.
c. Distribute the total sample evenly across the width of the well using a 250-µL Hamilton
microsyringe. Limited mixing of sample and electrophoresis buffer will facilitate even
loading and is easily dealt with by the strong stacking capability of the gel system
(see Note 11).
24. Fill the upper buffer reservoir with electrophoresis buffer.
25. Attach the electrical leads between power supply and electrophoresis system: the + lead to
the upper and the - lead to the lower reservoir. Note that this is opposite to the SDS gel
electrophoresis configuration. Remember, basic proteins such as histones are positively
charged and will move toward the cathode (negative electrode).